Immune complexes are formed by the binding of antibodies, consisting of various types of immunoglobulins, with antigens. The production of antibodies and formation of immune complexes is part of the immune response of an individual to combat disease.
Many diseases of autoimmune, infectious, or malignant nature are characterized in part by the presence of immune complexes in the blood. These complexes may remain in the circulation for long periods of time and deposit in various tissues, such as the lung, kidney, and heart, contributing to the inflammatory and erosive manifestations of autoimmune and other diseases. [See Emancipator, et al., Lab. Invest., 54:475-478 (1986)]. Prolonged presence of immune complexes in the circulation and in tissues contributes to a compromised immune system function and inflammatory pathology.
Immune complexes may be cleared from the circulation and from tissues by a variety of mechanisms. One naturally-occurring mechanism is the acute phase response. An acute phase response is the group of metabolic and systemic physiological changes an organism undergoes following a trauma, such as an infection, injury, or disease. At the tissue level, this response manifests itself as inflammation. At the organismal level, this response is characterized by changes in cellular populations and their functions. For example, circulating macrophages and fixed macrophages found in the liver, spleen, and lymph nodes, may remove immune complexes.
The acute phase response is also characterized by changes in concentrations of various metals and proteins in the circulation. Serum amyloid P component ("SAP") and C-reactive protein ("CRP") are examples of two serum proteins, the concentrations of which can increase rapidly and dramatically during an acute phase response. In humans undergoing acute phase responses, serum concentrations of SAP may fluctuate but do not rise significantly. In contrast, CRP serum concentrations can increase 1000-fold in humans during an acute phase response. In other species, however, SAP serum concentrations do increase. For example, murine SAP concentrations increase up to 50-fold during an induced acute phase response. Accordingly, SAP is considered an acute phase reactant ("APR") in mice but not in humans. [See Kushner, I., Ann. N.Y. Acad. Sci., 389:39-45 (1982); Pepys, M. B., et al., Nature, 278:199-201 (1979)].
Another mechanism by which immune complexes may be removed from the circulation is through plasma exchange or blood filtration therapy. Generally, for blood filtration therapy, a substance which can bind aggregated or complexed immunoglobulin is immobilized on a solid support which is encased in an extracorporeal device, through which whole blood or plasma is passed. In some cases, after passage over the adsorbent, blood components are reinfused into the individual thus eliminating the need for large amounts of replacement plasma or other replacement fluids. Published studies indicate that the removal of circulating immune complexes, or enhancing their clearance from the circulation, may constitute an effective therapeutic treatment. [See Theofilopoulos, A. M., et al., Adv. Immunol., 28:90-220 (1979); Theofilopolous, A. M., et al., Immunodiaonostics of Cancer, p. 896 (M. Decker Inc., New York 1979)].
Many investigators have devised assays and adsorbents to selectively react with immune complexes as a means of monitoring and removing immune complexes from fluids. Various methods have been used to assay for immune complexes, including the following: physical separation using polyethylene glycol; reducing the temperature of the solution containing the immune complexes (i.e., cold precipitation); binding of the immune complexes to complement protein C1q or to antibodies specific to the complement protein C3 and C3 degradation products; binding of the immune complexes to rheumatoid factors; binding to the bovine protein conglutinin; or binding of the immune complexes to platelets or to the Raji lymphoblastoid cell line. [See Theofilopoulos, A. M., et al., Hosp. Pract., pp. 107-121 (February 1980); Ingram, Animal Models of Immunological Processes, pp. 221-253 (1982); Levinsson, et al., J. Clin. Immunol., 7:328-336 (1984); Singh, et al., J. Immunol. Meth., 50:109-114 (1982); Hay, et al., Clin. Exp. Immunol., 24:396-400 (1976); Pereira, et al., J. Immunol., 125:763-770 (1980); Theofilopoulos, et al., J. Clin. Invest., 61:1570 (1978); Creighton, et al., J. Immunol., 111:1219 (1973); Schur, N. Engl. J. Med., 298:161 (1978); Bruneau, et al., J. Clin. Invest., 64:191 (1979)].
There are advantages to each of the aforementioned methods, as well as disadvantages which include insensitivity, nonspecificity, inability to detect immune complexes of all sizes and of all immunoglobulin isotypes and subisotypes, reliance on immune complexes containing complement proteins, and interference by non-complexed immunoglobulins. Therefore, results gathered by any of these methods and assays may be considered to be of marginal value for diagnostic purposes.
Other methods have been developed in which various adsorbents, immobilized on solid supports, are used for binding immune complexes. The use of Staphylococcal A (the various subtypes collectively known as "Protein A") as an immobilized immune complex adsorbent has had mixed results. The failure of Protein A as an adsorbent is presumably due to its inability to effectively differentiate the aggregated immunoglobulin of immune complexes from "noncomplexed" immunoglobulin. [See Betram, et al., J. Biol. Resp. Mod., 2:235-240 (1984); Terman, et al., New Engl. J. Med., 305:1195-2000 (1981); Dobre, et al., J. Immunol. Meth., 66:171-178 (1984); Nilsson, et al., Scand. J. Haematol., 30:458-464 (1983); Nauts, Host Defense Against Cancer and Its Potentiation, pp. 337-351 (1975)]. Because protein A binds to the "Fc" portion of immunoglobulins, investigators have also proposed or attempted using other Fc receptors, such as C1q or rheumatoid factor, or a specific antigen or antibody, as immobilized immunoadsorbents. [See Nilsson, I. M., et al., Plasma. Ther. Transfers. Technol., 5:127-134 (1984); Lai, K. N., et al., Artificial Organs, 11:259-264 (1987), Nilsson, I. M., et al., In Factor VIII Inhibitors, p. 225 (1984); Liberti, P. A., et al., J. Immunol., 123:2212-2219 (1979); Randerson, D. H., et al., Artificial Organs, 6:43-49 (1982)].
As described above, CRP is a protein whose serum concentration is known to significantly increase in humans during an acute phase response, and the CRP molecule has been monitored as a marker of acute inflammation. [See Potempa, L. A., et al., Mol. Immunol., 20:1165-1175 (1983)]. CRP can also recognize and bind one of several ligands on the cell or bacterial surface, or in suspension. These ligands include phosphorylcholine, chromatin, and polycations. It appears that certain CRP-ligand complexes have the capacity to activate the complement pathway, thus stimulating certain aspects of the immune system.
The native form of CRP can be altered so as to have significantly different charge, size, solubility, and antigenicity characteristics. The distinctive antigenicity associated with altered CRP has been referred to as "neo-CRP," and the altered CRP molecule itself has been referred to as "modified-CRP." It has been determined that modified-CRP expressing neo-CRP antigenicity functions in a variety of in vitro assays which are useful in assessing the state of immune system reactivity. [See Potempa, L. A., et al., Protides of the Biological Fluids, 34:287-290 (1986); Potempa, L. A., et al., Inflammation, 12:391-405 (1988); Gupta, R. C., et al., Arthritis & Rheumatism, 31:R39a (1988); Chu, E. B., et al., Amer. Assoc. Cancer Res. 29:371a (1988); Doughery, T. J., et al., Protides of the Biological Fluids, 34:291-293 (1986)].
Modified-CRP has also been used in vivo. Mice injected with modified-CRP 30 minutes prior to receiving a lethal dose (90%) of type 7F Streptococcus pneumoniae survived death in a significant and dose-related manner. [See Chudwin, D. S., et al., J. Allergy Clin. Immunol., 77:2169 (1986)].
Finally, as disclosed in PCT application WO 89/09628, modified-CRP, either in solution or immobilized on a solid surface, selectively binds aggregated immunoglobulin and immune complexes. This patent application describes the use of modified-CRP to detect and quantitate immune complexes and to remove aggregated immunoglobulin and immune complexes from fluids such as plasma. Also described is a method of reducing the levels of immune complexes in a mammal comprising administering modified-CRP to the mammal.
Serum amyloid P component ("SAP") is a naturally-occurring serum protein in mammals. SAP is a 250,000 molecular weight glycoprotein and is a member of the pentraxin family of structurally related proteins. [See Osmand, A. P., et al., Proc. Nat. Acad. Sci., 74:739-743 (1977)]. It has been hypothesized that the genes encoding SAP and CRP arose from a gene duplication since the genes for both SAP and CRP have been mapped to the same human chromosome, and have been found to be partially, but significantly, structurally homologous. SAP has approximately 50% strict amino acid sequence homology with CRP. [See Floyd-Smith, G., et al., Immunogenetics, 24:171-176 (1986); Mantzousanis, E. C., et al., J. Biol. Chem., 260:7752-7756 (1985); Ohnishi, S., J. Biol. Chem., 100:849-858 (1986); Prelli, F., et al., J. Bio. Chem., 260:12895-12898 (1985)].
The SAP molecule itself is quite different from CRP structurally. SAP circulates as two non-covalently associated pentamers, each of which consists of five identical non-covalently linked subunits. [See Painter, R. H., Ann. N.Y. Acad. Sci., 389:199-215 (1982); Perkins, S. J., et al., Protides of the Biological Fluids, 34:323-326 (1986)]. While SAP exists as a decameric molecule in solution, CRP behaves as a pentamer in coexistence with higher oligomers. Id.
SAP aggregates and precipitates in high concentrations of ionic calcium. It has been suggested that SAP may aggregate and precipitate to form amyloid P component ("AP") in localized areas of increased ionic calcium concentration. [See Baltz, M. L., Biochim. Biophys. Acta, 701:229-236 (1982)]. AP is a normal constituent of glomerular basement membrane, human dermis, and cervix, testis, and placentae tissues. [See Baltz, M. L., et al., Clin. Exp. Immunol., 66:691-700 (1986); Dyck, R. F., et al., J. Exp. Med., 152:1162-1174 (1980); Melvin, T., Am. J. Pathol., 125:460-464 (1986); Breathnach, S. M., J. Invest. Derm., 92:53-58 (1989); Clayton, J., Cell. Pathol., 43:63-66 (1983); Herriut, R., et al., J. Pathol., 157:11-14 (1989); Khan, A. M., et al., Placenta, 6:551-554 (1985)].
SAP has binding interactions with both cells and ligands. Specifically, with respect to cellular binding, SAP has been shown to bind to elicited, but not quiescent, tissue macrophages, thereby exerting a strong, positive feedback affect on interleukin-1 elaboration. [See Sarlo, K. T., Cell. Immunol., 93:398-405 (1985)]. Furthermore, SAP has been reported to inhibit CRP-induced platelet aggregation and to inhibit murine secondary antibody responses to T-cell dependent antigens in vitro. [See Fiedel, B. A., et al., J. Immunol., 131:1416-1419 (1983); Sarlo, K. T., et al., Cell. Immun., 106:273-286 (1987)].
The binding of SAP to ligands has also been demonstrated. Ligands of SAP include unsubstituted agarose, heparin, isolated amyloid fibrils, zymosan, fibronectin, C3bi, C4bp, C1q, and the Fab fragment of IgG. [See Painter, R. H., et al., Ann. N.Y. Acad. Sci., 389:199-215 (1982); Boxer, G. J., et al., Blood, 50:260 (Abstr.) (1977); Pepys, M. B., et al., Clin. Exp. Immunol., 38:284-293 (1979); Deeber, F. C., et al., J. Exp. Med., 154:1134-1149 (1981); Tseng, J., et al., Immunol. Invest., 15:749-761 (1986); Rostagno, A., et al., B.B.R.C., 140:12-20 (1986); Hutchcraft, C., Ann. N.Y. Acad. Sci., 389:449-450 (1982); Bristow, C. L., et al., Mol. Immunol., 23:1045-1052 (1986)]. This binding has consistently shown to be calcium-dependent.
Also shown to be specifically bound by SAP in vitro have been various, but not all, glycosaminoglycans, immobilized native and denatured DNA, native chromatin, and H1-deficient chromatin. [See Hamazaki, H., J. Biol. Chem., 262:1456-1460 (1987); Pepys, M. B., et al., B.B.R.C., 148:308-313 (1987)].
The nature of the specific binding interactions between SAP and some of its cellular, tissue-bound, and soluble ligands has been investigated. The results of an investigation into the structure of pyruvate-rich agarose components to which SAP specifically and avidly binds suggest that a glycan structurally similar to calcium sequestering .beta.-turns connecting antiparallel .beta.-pleated sheaths in proteins were the target of SAP interactions. It has been proposed that .beta.-pleated sheath secondary structure binds to SAP and becomes the associated AP component always found in these tissues. [See Hind, C. R., et al., J. Exp. Med., 159:1058-1069 (1984)]. Galactans, but not galactose, competed with nonT, nonB ALL cells and peripheral blood mononuclear cells for labeled AP. Moreover, AP could be precipitated by two snail galactans in a dose-dependent manner. This suggests that appropriate conformation of a carbohydrate moiety is essential for SAP's binding and is supported by the finding that a human serum lectin (which was later identified as SAP) binds to the penultimate galactose residues in oligosaccharides but not well to terminal galactose residues, and not at all to fucose, N-acetylglucosamine, and mannose. [See Li, J. J., et al., Scand. J. Immunol., 19:227-236 (1984); Hamazaki, H., J. Biol. Chem., 261: 5455-5459 (1986); Hamazaki, H., B.B.R.C., 150:212-218 (1988)]. However, SAP has been found to bind mannose-rich oligosaccharide sequences and mannose-terminated sequences. [See Kubak, B. M., et al., Mol. Immunol., 25: 851-858 (1988)].
In a further effort to characterize SAP binding, SAP's binding to a panel of haptens was assessed. The results of these studies suggest that SAP has a single type of binding site which is capable of specifically binding a variety of ligands which share a single, as yet unidentified, characteristic. A model to explain this polyspecific binding by a single type of site is proposed in which SAP binds a calcium ion in such a way that it occupies only half of the ion's coordination sphere, the other half of which is then bound by SAP's ligand. In the absence of an exogenous ligand, it is proposed that the other half of the SAP-bound ionic calcium's coordination sphere is bound by other SAP binding sites within another, or the same, SAP molecule. This would explain the observed calcium-induced precipitation of SAP. Similar models can be invoked to explain the observation that copper can also induce SAP autoaggregation, as well as zymosan binding, under neutral and acidic conditions. [See Potempa, L. A., et al., J. Biol. Chem., 260:12142-12147 (1985)].
The same panel of haptens has been used to study the calcium-dependent ligand binding characteristics of CRP. CRP was found to bind only two of the ligands tested, and the binding was poor. The very limited interaction of CRP with the panel of SAP ligands was interpreted as an indication that CRP-bound ionic calcium cannot form calcium bridges as can SAP-bound ionic calcium. [See Serban, D., et al., Scan. J. Immunol., 25:275-281 (1987)].
The nature of the binding of fibronectin and elicited macrophages by SAP was analyzed by Scatchard analysis of competitive ELISA and radiolabeled-SAP binding assays. A Scatchard plot depicting the results of an ELISA in which the binding of soluble fibronectin to immobilized SAP was measured indicated that positive cooperativity was involved. The binding of radiolabeled-SAP to elicited macrophages exhibited all of the hallmarks of receptor binding (e.g., specificity, saturability, reversibility, and high affinity). There appeared to be approximately 200,000 of a single class of macrophage receptors with an equilibrium dissociation constant of 5.times.10.sup.-8 M per cell. Based upon competition studies and studies of the affect of deglycosylating SAP on its binding, it was proposed that the elicited macrophage receptor to which SAP binds is a cation-dependent mannose-6-phosphate receptor. This is in contrast to the macrophage receptor for CRP which has been found to be closely associated with the IgG Fc receptor. [See Mortensen, R. F., et al., J. Immunol., 119:1611 (1977)].